A conformational change in the alpha-subunit of coatomer induced by ligand binding to gamma-COP revealed by single-pair FRET

Formation of transport vesicles involves polymerization of cytoplasmic coat proteins (COP). In COPI vesicle biogenesis, the heptameric complex coatomer is recruited to donor membranes by the interaction of multiple coatomer subunits with the budding machinery. Specific binding to the trunk domain of γ-COP by the Golgi membrane protein p23 induces a conformational change that causes polymerization of the complex. Using single-pair fluorescence resonance energy transfer, we find that this conformational change takes place in individual coatomer complexes, independent of each other, and that the conformational rearrangement induced in γ-COP is transmitted within the complex to its α-subunit. We suggest that capture of membrane protein machinery triggers cage formation in the COPI system.     

Two human ARFGAPs associated with COP-I-coated vesicles

ADP-ribosylation factors (ARFs) are critical regulators of vesicular trafficking pathways and act at multiple intracellular sites. ADP-ribosylation factor-GTPase-activating proteins (ARFGAPs) are proposed to contribute to site-specific regulation. In yeast, two distinct proteins, Glo3p and Gcs1p, together provide overlapping, essential ARFGAP function required for coat protein (COP)-I-dependent trafficking. In mammalian cells, only the Gcs1p orthologue, named ARFGAP1, has been characterized in detail. However, Glo3p is known to make the stronger contribution to COP I traffic in yeast. Here, based on a conserved signature motif close to the carboxy terminus, we identify ARFGAP2 and ARFGAP3 as the human orthologues of yeast Glo3p. By immunofluorescence (IF), ARFGAP2 and ARFGAP3 are closely colocalized with coatomer subunits in NRK cells in the Golgi complex and peripheral punctate structures. In contrast to ARFGAP1, both ARFGAP2 and ARFGAP3 are associated with COP-I-coated vesicles generated from Golgi membranes in the presence of GTP-gamma-S in vitro. ARFGAP2 lacking its zinc finger domain directly binds to coatomer. Expression of this truncated mutant (DeltaN-ARFGAP2) inhibits COP-I-dependent Golgi-to-endoplasmic reticulum transport of cholera toxin (CTX-K63) in vivo. Silencing of ARFGAP1 or a combination of ARFGAP2 and ARFGAP3 in HeLa cells does not decrease cell viability. However, silencing all three ARFGAPs causes cell death. Our data provide strong evidence that ARFGAP2 and ARFGAP3 function in COP I traffic.     

ARFGAP2 and ARFGAP3 Are Essential for COPI Coat Assembly on the Golgi Membrane of Living Cells

Coat protein complex I (COPI) vesicles play a central role in the recycling of proteins in the early secretory pathway and transport of proteins within the Golgi stack. Vesicle formation is initiated by the exchange of GDP for GTP on ARF1 (ADP-ribosylation factor 1), which, in turn, recruits the coat protein coatomer to the membrane for selection of cargo and membrane deformation. ARFGAP1 (ARF1 GTPase-activating protein 1) regulates the dynamic cycling of ARF1 on the membrane that results in both cargo concentration and uncoating for the generation of a fusion-competent vesicle. Two human orthologues of the yeast ARFGAP Glo3p, termed ARFGAP2 and ARFGAP3, have been demonstrated to be present on COPI vesicles generated in vitro in the presence of guanosine 5′-3-O-(thio)triphosphate. Here, we investigate the function of these two proteins in living cells and compare it with that of ARFGAP1. We find that ARFGAP2 and ARFGAP3 follow the dynamic behavior of coatomer upon stimulation of vesicle budding in vivo more closely than does ARFGAP1. Electron microscopy of ARFGAP2 and ARFGAP3 knockdowns indicated Golgi unstacking and cisternal shortening similarly to conditions where vesicle uncoating was blocked. Furthermore, the knockdown of both ARFGAP2 and ARFGAP3 prevents proper assembly of the COPI coat lattice for which ARFGAP1 does not seem to play a major role. This suggests that ARFGAP2 and ARFGAP3 are key components of the COPI coat lattice and are necessary for proper vesicle formation.     

Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression. However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions between δ and ε and δ and γ subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.     

Using yeast two-hybrid system to detect interactions of ATP synthase subunits fromSpinacia oleracea

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression. However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions between δ and ε and δ and γ subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.     

The structure and function of the beta 2-adaptin appendage domain

The heterotetrameric AP2 adaptor (alpha, beta 2, mu 2 and sigma 2 subunits) plays a central role in clathrin-mediated endocytosis. We present the protein recruitment function and 1.7 A resolution structure of its beta 2-appendage domain to complement those previously determined for the mu 2 subunit and alpha appendage. Using structure-directed mutagenesis, we demonstrate the ability of the beta 2 appendage alone to bind directly to clathrin and the accessory proteins AP180, epsin and eps15 at the same site. Clathrin polymerization is promoted by binding of clathrin simultaneously to the beta 2-appendage site and to a second site on the adjacent beta 2 hinge. This results in the displacement of the other ligands from the beta 2 appendage. Thus clathrin binding to an AP2-accessory protein complex would cause the controlled release of accessory proteins at sites of vesicle formation.     

Solution Structure of Human ζ-COP: Direct Evidences for Structural Similarity between COP I and Clathrin-Adaptor Coats

COP-I-coated vesicles are protein and lipid carriers that mediate intra-Golgi transport and transport from the cis-Golgi complex to the endoplasmic reticulum in cells. The coatomer of the vesicles coat is comprised of seven subunits: α-COP, ?-COP, β′-COP, β-COP, γ-COP, δ-COP, and ζ-COP. Here we report the solution structure of a truncated form (residues 1-149; ζ-COP149) of human ζ-COP (total 177 residues). It is the first three-dimensional structure of a “core” subunit of the COP I F-subcomplex. The structure of ζ-COP149 mainly consists of a disordered N-terminal tail, a five-stranded antiparallel β-sheet, a two-stranded antiparallel β-sheet, and five α-helices. The global folding of ζ-COP149 is very similar to the crystal structures of AP1-σ1 and AP2-σ2, directly demonstrating the structural similarity between the “core” subunits of the COP I F-subcomplex and adaptor protein complexes. Through structural comparison and mutagenesis study, we have also demonstrated that the heterodimers of ζ-COP149 and γ-COP have packing interfaces and relative subunit orientations similar to those of AP2-σ2 and AP2-α heterodimers. These results provide direct evidence supporting the previous proposal that the COP I F-subcomplex and adaptor protein complexes have similar tertiary and quaternary structures.     

Physiological Functions of the COPI Complex in Higher Plants

COPI vesicles are essential to the retrograde transport of proteins in the early secretory pathway. The COPI coatomer complex consists of seven subunits, termed α-, β-, β′-, γ-, δ-, ε-, and ζ-COP, in yeast and mammals. Plant genomes have homologs of these subunits, but the essentiality of their cellular functions has hampered the functional characterization of the subunit genes in plants. Here we have employed virus-induced gene silencing (VIGS) and dexamethasone (DEX)-inducible RNAi of the COPI subunit genes to study the in vivo functions of the COPI coatomer complex in plants. The β′-, γ-, and δ-COP subunits localized to the Golgi as GFP-fusion proteins and interacted with each other in the Golgi. Silencing of β′-, γ-, and δ-COP by VIGS resulted in growth arrest and acute plant death in Nicotiana benthamiana, with the affected leaf cells exhibiting morphological markers of programmed cell death. Depletion of the COPI subunits resulted in disruption of the Golgi structure and accumulation of autolysosome-like structures in earlier stages of gene silencing. In tobacco BY-2 cells, DEX-inducible RNAi of β′-COP caused aberrant cell plate formation during cytokinesis. Collectively, these results suggest that COPI vesicles are essential to plant growth and survival by maintaining the Golgi apparatus and modulating cell plate formation.     

Silkworm coatomers and their role in tube expansion of posterior silkgland

Background

Coat protein complex I (COPI) vesicles, coated by seven coatomer subunits, are mainly responsible for Golgi-to-ER transport. Silkworm posterior silkgland (PSG), a highly differentiated secretory tissue, secretes fibroin for silk production, but many physiological processes in the PSG cells await further investigation.

Methodology/Principal Findings

Here, to investigate the role of silkworm COPI, we cloned six silkworm COPI subunits (α,β,β′, δ, ε, and ζ-COP), determined their peak expression in day 2 in fifth-instar PSG, and visualized the localization of COPI, as a coat complex, with cis-Golgi. By dsRNA injection into silkworm larvae, we suppressed the expression of α-, β′- and γ-COP, and demonstrated that COPI subunits were required for PSG tube expansion. Knockdown of α-COP disrupted the integrity of Golgi apparatus and led to a narrower glandular lumen of the PSG, suggesting that silkworm COPI is essential for PSG tube expansion.

Conclusions/Significance

The initial characterization reveals the essential roles of silkworm COPI in PSG. Although silkworm COPI resembles the previously characterized coatomers in other organisms, some surprising findings require further investigation. Therefore, our results suggest the silkworm as a model for studying intracellular transport, and would facilitate the establishment of silkworm PSG as an efficient bioreactor.     

Molecular and Pharmacological Characterization of GABAA Receptors in the Rat Pituitary

Abstract: Levels of mRNA for the major subunits of the GABA_A receptor were assayed in the rat pituitary anterior and neurointermediate lobes by ribonuclease protection assay. α1, β1, β2, β3, and γ2s were found to be the predominant subunits in the anterior lobe, whereas α2, α3, β1, β3, γ2s, and γ1 were the predominant subunits expressed in the neurointermediate lobe. α5, α6, and δ subunits were not detectable. Hill and Scatchard analysis of [³H]muscimol binding to anterior and neurointermediate lobe membranes showed high-affinity binding sites with dissociation constants of 5.6 and 4.5 n M , respectively, and Hill coefficients near 1. Muscimol sites were present at a maximum of 126 fmol/mg in the anterior lobe and 138 fmol/mg in the neurointermediate lobe. The central-type benzodiazepine antagonist [³H]Ro 15-1788 bound to a high-affinity site with a dissociation constant of 1.5 n M in both tissues, at a maximum of 60 fmol/mg in anterior pituitary and 72 fmol/mg in neurointermediate lobe. A Hill coefficient of 1 was measured for this site in both tissues. Assays of CL 218 872 displacement of Ro 15-1788 were consistent with a pure type I benzodiazepine site in the anterior lobe and a pure type II site in the intermediate lobe. These results are consistent with both tissue-specific expression of particular GABA_A receptor subunits and receptor heterogeneity within individual cells in the pituitary.     

Conserved structural motifs in intracellular trafficking pathways: structure of the gammaCOP appendage domain

The formation of coated vesicles is a fundamental step in many intracellular trafficking pathways. COPI and clathrin represent two important and distinct sets of vesicle coating machinery, involved primarily in mediating intra-Golgi and endocytic transport, respectively. Here we identify an important functional region at the carboxyl terminus of the gamma subunit of the COPI complex (gammaCOP) and describe the X-ray crystal structure of this domain at 2.3 A resolution. This domain of gammaCOP exhibits unexpected structural similarity to the carboxyl-terminal appendage domains of the alpha and beta subunits of the AP2 adaptor proteins, integral components of clathrin-coated vesicles. The remarkable structural conservation exhibited by the gammaCOP appendage domain, coupled with functional data and primary sequence analysis, supports a model of COPI function with significant structural and mechanistic parallels to vesicular transport by the clathrin/AP2 system.     

Clathrin interactions with C-terminal regions of the yeast AP-1 beta and gamma subunits are important for AP-1 association with clathrin coats

Heterotetrameric adaptor (AP) complexes are thought to coordinate cargo recruitment and clathrin assembly during clathrin-coated vesicle biogenesis. We have identified, and characterized the physiological significance of clathrin-binding activities in the two large subunits of the AP-1 complex in Saccharomyces cerevisiae . Using GST-fusion chromatography, two clathrin-binding sites were defined in the β1 subunit that match consensus clathrin-binding sequences in other mammalian and yeast clathrin-binding proteins. Clathrin interactions were also identified with the C-terminal region of the γ subunit. When introduced into chromosomal genes, point mutations in the β1 clathrin-binding motifs, or deletion of the γ C-terminal region, reduced association of AP-1 with clathrin in coimmunoprecipitation assays. The β1 mutations or the γ truncation individually produced minor effects on AP-1 distribution by subcellular fractionation. However, when β1 and γ mutations were combined, severe defects were observed in AP-1 association with membranes and incorporation into clathrin-coated vesicles. The combination of subunit mutations accentuated growth and α-factor pheromone maturation defects in chc1-ts cells, though not to the extent caused by complete loss of AP-1 activity. Our results suggest that both the β1 and γ subunits contribute interactions with clathrin that are important for stable assembly of AP-1 complexes into clathrin coats in vivo .     

Effects of mutations in the WD40 domain of α-COP on its interaction with the COPI coatomer in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Replacement of glycine 227 in the fifth WD40 motif of α-COP/Ret1p/Soo1p by charged or aromatic amino acids is responsible for the temperature-dependent osmo-sensitivity of Saccharomyces cerevisiae, while truncations of WD40 motifs exerted a reduction in cell growth rate and impairment in assembly of cell-wall associated proteins such as enolase and Gas1p. Yeast two-hybrid analysis revealed that the ret1-1/soo1-1 mutation of α-COP abolished the interaction with β- and ɛ-COP, respectively, and that the interaction between α-COP and β-COP relied on the WD40 domain of α-COP. Furthermore, although the WD40 domain is dispensable for interaction of α-COP with ɛ-COP, structural alterations in the WD40 domain could impair the interaction.     

Phylogenetic Relationships of the Seven Coat Protein Subunits of the Coatomer Complex, and Comparative Sequence Analysis of Murine Xenin and Proxenin

The coatomer complex is involved in intracellular protein transport and comprises an assembly of seven polypeptide subunits designated , , , , , , and COP. Rooted phylogenetic trees constructed from the full-length cDNA and amino acid sequences of 49 COP entities in different eukaryotes from yeast to man generally revealed striking conservation of each subunit through evolution. Both nucleotide and protein trees displayed close relationships between and subunits, between and subunits, and between and subunits, implying evolution from common ancestors as well as functional similarity. Interestingly, although 6 out of 7 -COP genes appeared to be grouped and related to the -COP genes, 4 out of 7 -COP gene products clustered with other groups of other COP subunit proteins. A 5 coding segment of the murine -COP gene was amplified by RT-PCR and cycle-sequenced. The partial predicted amino acid sequence of this murine homolog was exactly identical to the human and bovine counterparts. Of particular significance was the complete identity of the first 25 and 35 N-terminal residues which constitute the gastrointestinal hormone xenin and its precursor proxenin, thus emphasizing their strict evolutionary conservation and alluding to their physiological importance.     

KDEL-cargo regulates interactions between proteins involved in COPI vesicle traffic: measurements in living cells using FRET.

How the occupied KDEL receptor ERD2 is sorted into COPI vesicles for Golgi-to-ER transport is largely unknown. Here, interactions between proteins of the COPI transport machinery occurring during a "wave" of transport of a KDEL ligand were studied in living cells. FRET between CFP and YFP fusion proteins was measured by multifocal multiphoton microscopy and bulk-cell spectrofluorimetry. Ligand binding induces oligomerization of ERD2 and recruitment of ARFGAP to the Golgi, where the (ERD2)n/ARFGAP complex interacts with membrane-bound ARF1. During KDEL ligand transport, interactions of ERD2 with beta-COP and p23 decrease and the proteins segregate. Both p24a and p23 interact with ARF1, but only p24 interacts with ARFGAP. These findings suggest a model for how cargo-induced oligomerization of ERD2 regulates its sorting into COPI-coated buds.     

Sequences in the Amino Termini of GABA ρ and GABAA Subunits Specify Their Selective Interaction In Vitro

Abstract: Molecular cloning has revealed that there are six classes of subunits capable of forming GABA-gated chloride channel receptors. GABA_A receptors are composed of α, β, γ, δ, and ε/χ subunits, whereas GABA_C receptors appear to contain ρ subunits. However, retinal cells exhibiting GABA_C responses express α, β, and ρ subunits, raising the possibility that GABA_C receptors may be a mixture of subunit classes. Using in vitro translated protein, we determined that human GABA_A receptor subunits α1, α5, and β1 did not coimmunoprecipitate with full-length ρ1, ρ2, or the N-terminal domain of ρ1 that contains signals for ρ-subunit interaction. To explore the molecular mechanism underlying these apparently exclusive combinations, chimeric subunits were created and tested for interaction with the wild-type subunits. Transfer of the N terminus of β1 to ρ1 created a β1ρ1 chimera that coimmunoprecipitated with the α1 subunit but not with the ρ2 subunit. Furthermore, exchanging the N terminus of the ρ1 subunit with the corresponding region of β1 produced a ρ1β1 chimera that interfered with ρ1 receptor expression in Xenopus oocytes, whereas the full-length β1 subunit had no effect. Together, these results indicate that sequences in the N termini direct assembly of ρ subunits and GABA_A subunits into GABA_C and GABA_A receptors, respectively.     

The α5(H105R) mutation impairs α5 selective binding properties by altered positioning of the α5 subunit in GABAA receptors containing two distinct types of α subunits

GABA_A receptors are pentameric ligand-gated ion channels that are major mediators of fast inhibitory neurotransmission. Clinically relevant GABA_A receptor subtypes are assembled from α5(1-3, 5), β1-3 and the γ2 subunit. They exhibit a stoichiometry of two α, two β and one γ subunit, with two GABA binding sites located at the α/β and one benzodiazepine binding site located at the α/γ subunit interface. Introduction of the H105R point mutation into the α5 subunit, to render α5 subunit-containing receptors insensitive to the clinically important benzodiazepine site agonist diazepam, unexpectedly resulted in a reduced level of α5 subunit protein in α5(H105R) mice. In this study, we show that the α5(H105R) mutation did not affect cell surface expression and targeting of the receptors or their assembly into macromolecular receptor complexes but resulted in a severe reduction of α5-selective ligand binding. Immunoprecipitation studies suggest that the diminished α5-selective binding is presumably due to a repositioning of the α5(H105R) subunit in GABA_A receptor complexes containing two different α subunits. These findings imply an important role of histidine 105 in determining the position of the α5 subunit within the receptor complex by determining the affinity for assembly with the γ2 subunit.     

Mutational analysis of βCOP (Sec26p) identifies an appendage domain critical for function

Background  

The appendage domain of the γCOP subunit of the COPI vesicle coat bears a striking structural resemblance to adaptin-family appendages despite limited primary sequence homology. Both the γCOP appendage domain and an equivalent region on βCOP contain the FxxxW motif; the conservation of this motif suggested the existence of a functional appendage domain in βCOP.     

ARFGAP1 promotes AP-2-dependent endocytosis

COPI (coat protein I) and the clathrin-AP-2 (adaptor protein 2) complex are well-characterized coat proteins, but a component that is common to these two coats has not been identified. The GTPase-activating protein (GAP) for ADP-ribosylation factor 1 (ARF1), ARFGAP1, is a known component of the COPI complex. Here, we show that distinct regions of ARFGAP1 interact with AP-2 and coatomer (components of the COPI complex). Selectively disrupting the interaction of ARFGAP1 with either of these two coat proteins leads to selective inhibition in the corresponding transport pathway. The role of ARFGAP1 in AP-2-regulated endocytosis has mechanistic parallels with its roles in COPI transport, as both its GAP activity and coat function contribute to promoting AP-2 transport.     

Rab2 requires PKC iota/lambda to recruit beta-COP for vesicle formation

The small GTPase Rab2 initiates the recruitment of soluble components necessary for protein sorting and recycling from pre-Golgi intermediates. Our previous studies showed that Rab2 required protein kinase C (PKC) or a PKC-like protein to recruit β-COP to membrane (Tisdale EJ, Jackson M. Rab2 protein enhances coatomer recruitment to pre-Golgi intermediates. J Biol Chem 1998;273: 17269–17277). We investigated the role of PKC in Rab2 function by first determining the active isoform that associates with membranes used in our assay. Western blot analysis detected three isoforms: PKCα, Γ and ι/Λ. A quantitative binding assay was used to measure recruitment of these kinases when incubated with Rab2. Only PKCι/Λ translocated to membrane in a dose-dependent manner. Microsomes treated with anti-PKCι/Λ lost the ability to bind β-COP, suggesting that Rab2 requires PKCι/Λ for β-COP recruitment. The recruitment of β-COP to membranes is not regulated by PKCι/Λ kinase activity. However, PKCι/Λ kinase activity was necessary for Rab2-mediated vesicle budding. We found that the addition of either a kinase-deficient PKCι/Λ mutant or atypical PKC pseudosubstrate peptide to the binding assay drastically reduced vesicle formation. These data suggest that Rab2 causes translocation of PKCι/Λ to v ¯esicular t ¯ubular c ¯lusters (VTCs), which promotes the recruitment of COPI to generate retrograde-transport vesicles.